PROJECT SUMMARY/ABSTRACT The CRISPR/Cas9 system has quickly emerged as the most robust gene editing tool yet discovered. Many early papers employing this system have utilized a plasmid that encodes the Cas9 protein and the guide RNA (sgRNA). These plasmids are typically delivered in viral shells that can often be too small for the entire plasmid, requiring multiple segments to be transported separately. Another drawback of plasmid incorporation is the continual expression of the protein and RNA, which often leads to undesired off-target effects. A promising alternative is the delivery of the Cas9sgRNA complex directly. A variety of methods have been developed for the delivery of this complex, including lipid nanoparticles and cell-penetrating peptides, but all methods so far result in inefficient gene editing or are inapplicable in vivo (or both). Recent work in our lab has demonstrated that the unique chemistry of the benzoxaborole functional group allows it to deliver proteins efficiently and directly into the cytosol. Combining this unique chemistry with the reactivity of a diazo motif will allow us to develop small molecules that can mask Cas9 carboxylates via an esterification reaction. A series of these delivery vehicles will be synthesized and reacted with the Cas9sgRNA complex to evaluate their effect on complex stability and protein?RNA binding. Initial experiments will focus on knocking out the GFP gene in GFP-producing HEK cells. Once the optimal delivery vehicle and conditions have been determined, that vehicle will be employed as a tool in future studies of breast cancer metastasis. Collagen prolyl 4-hydroxylase has been demonstrated to be significant in the metastasis process, and we anticipate that knock-out of this gene via a CRISPR/Cas9 system would be an effective approach. Finally, our delivery strategy could provide a straightforward method for delivering not only Cas9sgRNA complexes, but also a wide variety of proteins.